Abstract The effort of much research has concentrated on the identification of autoantigens in type 1 diabetes (T1D) as targets for therapeutic interventions and as predictive biomarkers for disease risk. Our lab has recently discovered a unique class of post- translationally modified antigens that are the ligands for pathogenic CD4 T cell clones isolated from the NOD mouse, the predominant model for T1D. The antigens are comprised of fragments of proinsulin (C-peptide) covalently linked to other natural cleavage products of ?-cell secretory granule proteins to form ?hybrid insulin peptides? (HIPs). The identification of HIP-specific CD4 T cells cultured from the infiltrated pancreatic islets of T1D individuals implies that HIPs may also serve as putative epitopes for autoantibodies. The primary goal of this proposal is to identify a novel panel of autoantibody biomarkers reactive to HIP antigens among T1D individuals. Autoantibodies to HIPs could serve as early indicators of active ?-cell destruction and as parameters for stratification of candidates that would most benefit from immunotherapy. We hypothesize that HIPs and autoantibodies targeting human HIP epitopes can be detected in the bloodstream of newly diagnosed T1D and individuals at high risk for disease. We propose to develop sensitive and specific bioassays focusing on these new HIP antigens as targets. First, we will design radioimmunoprecipitation assays to detect autoantibodies in the sera of T1D patients targeting the known human HIPs. The assay conditions will be optimized and specificity demonstrated with competitive ligands. Concurrently we will explore the possibility that HIPs are directly detectable in the circulation as they may be co-secreted with insulin or be a casualty of ?-cell stress. Anti- HIP monoclonal antibodies will be used to capture HIPs from sera in a modified ELISA format. Finally, if our hypothesis that formation of HIPs plays a key role in the pathogenesis of T1D is valid, then a broader collection of HIPs has yet to be identified. We will screen human HIP libraries with T1D sera to identify humoral immunoreactivity to additional HIP sequences. HIPs or autoantibodies targeting HIPS would introduce a new generation of biomarkers in T1D translational research. They could provide metrics for disease risk, staging of ?-cell destruction and the foundation for HIP epitope targets for innovative drug development.